Mature mRNAs accumulated in the nucleus are neither the molecules in transit to the cytoplasm nor constitute a stockpile for gene expression

Weil, Dominique; Boutain, Sylvie; Audibert, Agnès; Dautry, François

doi:10.1017/s1355838200000479

Cited by 32 publications

(41 citation statements)

References 42 publications

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“…The Tet-regulated vector has been derived previously from the tTA expression vector (Dirks et al, 1994;Weil et al, 2000). The tTA/CPEB1-∆5-long plasmid was created by inserting a restriction fragment containing the full ORF of the pEGFP/CPEB1-∆5-long plasmid.…”

Section: Expression Vectorsmentioning

confidence: 99%

The translational regulator CPEB1 provides a link between dcp1 bodies and stress granules

Wilczynska

Aigueperse

Kress

et al. 2005

Journal of Cell Science

273

291

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The cytoplasmic polyadenylation element-binding protein (CPEB) has been characterized in Xenopus laevis as a translational regulator. During the early development, it behaves first as an inhibitor and later as an activator of translation. In mammals, its closest homologue is CPEB1 for which two isoforms, short and long, have been described. Here we describe an additional isoform with a different RNA recognition motif, which is differentially expressed in the brain and ovary. We show that all CPEB1 isoforms are found associated with two previously described cytoplasmic structures, stress granules and dcp1 bodies. This association requires the RNA binding ability of the protein, whereas the Aurora A phosphorylation site is dispensable. Interestingly, the rck/p54 DEAD box protein, which is known as a CPEB partner in Xenopus and clam, and as a component of dcp1 bodies in mammals, is also present in stress granules. Both stress granules and dcp1 bodies are involved in mRNA storage and/or degradation, although so far no link has been made between the two, in terms of neither morphology nor protein content. Here we show that transient CPEB1 expression induces the assembly of stress granules, which in turn recruit dcp1 bodies. This dynamic connection between the two structures sheds new light on the compartmentalization of mRNA metabolism in the cytoplasm.

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Section: Expression Vectorsmentioning

confidence: 99%

The translational regulator CPEB1 provides a link between dcp1 bodies and stress granules

Wilczynska

Aigueperse

Kress

et al. 2005

Journal of Cell Science

273

291

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“…Position of bands was determined following exposure to phosphor screens and scanning using a PhosphorImager. To test for the selective transport of particular env RNA forms, nuclear and cytoplasmic fractions were prepared as previously described (McCracken et al 1998) with the following amendments: Cells were harvested in 1× TBS, 2 mM EDTA and EDTA was added to the hypotonic lysis buffer to a final concentration of 10 mM to remove any remaining polysomal RNA from the nuclear fraction (Weil et al 2000). RNA was isolated and analyzed from the individual fractions as indicated above.…”

Section: A Novel Function For Sam68mentioning

confidence: 99%

A novel function for Sam68: Enhancement of HIV-1 RNA 3′ end processing

McLaren

Asai²,

Cochrane³

2004

RNA

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Both cis elements and host cell proteins can significantly affect HIV-1 RNA processing and viral gene expression. Previously, we determined that the exon splicing silencer (ESS3) within the terminal exon of HIV-1 not only reduces use of the adjacent 3 splice site but also prevents Rev-induced export of the unspliced viral RNA to the cytoplasm. In this report, we demonstrate that loss of unspliced viral RNA export is correlated with the inhibition of 3 end processing by the ESS3. Furthermore, we find that the host factor Sam68, a stimulator of HIV-1 protein expression, is able to reverse the block to viral RNA export mediated by the ESS3. The reversal is associated with a stimulation of 3 end processing of the unspliced viral RNA. Our findings identify a novel activity for the ESS3 and Sam68 in regulating HIV-1 RNA polyadenylation. Furthermore, the observations provide an explanation for how Sam68, an exclusively nuclear protein, modulates cytoplasmic utilization of the affected RNAs. Our finding that Sam68 is also able to enhance 3 end processing of a heterologous RNA raises the possibility that it may play a similar role in regulating host gene expression.

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“…RNA Preparation and Northern Blot-For preparation of nuclear and cytoplasmic RNA, 8 ϫ 10 6 cells were collected 48 h after transfection and treated according to the protocol of Weil et al (21). Briefly, the cells were resuspended in 500 l of Nonidet P-40 lysis buffer (0.5% Nonidet P-40, 0.14 M NaCl, 10 mM Tris, pH 8.4, 1.5 mM MgCl 2 , 10 mM EDTA, pH 8.0) for 5 min at 0°C.…”

Section: Methodsmentioning

confidence: 99%

Murine Leukemia Virus Regulates Alternative Splicing through Sequences Upstream of the 5· Splice Site

Kraunus

Zychlinski

Heise

et al. 2006

Journal of Biological Chemistry

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Alternative splicing of the primary transcript plays a key role in retroviral gene expression. In contrast to all known mechanisms that mediate alternative splicing in retroviruses, we found that in murine leukemia virus, distinct elements located upstream of the 5 splice site either inhibited or activated splicing of the genomic RNA. Detailed analysis of the first untranslated exon showed that the primer binding site (PBS) activates splicing, whereas flanking sequences either downstream or upstream of the PBS are inhibitory. This new function of the PBS was independent of its orientation and primer binding but associated with a particular destabilizing role in a proposed secondary structure. On the contrary, all sequences surrounding the PBS that are involved in stem formation of the first exon were found to suppress splicing. Targeted mutations that destabilized the central stem and compensatory mutations of the counter strand clearly validated the concept that murine leukemia virus attenuates its 5 splice site by forming an inhibitory stem-loop in its first exon. Importantly, this mode of splice regulation was conserved in a complete proviral clone. Some of the mutants that increase splicing revealed an opposite effect on translation, implying that the first exon also regulates this process. Together, these findings suggest that sequences upstream of the 5 splice site play an important role in splice regulation of simple retroviruses, directly or indirectly attenuating the efficiency of splicing.A characteristic feature of all retroviruses is the process of reverse transcription of the RNA genome into double-stranded DNA. Following integration into the host genome, the proviral DNA functions as one expression unit, which is transcribed by cellular RNA polymerase II, yielding a single polycistronic primary transcript that serves as genomic RNA for progeny virus. Productive infection and formation of new retroviral particles require the well balanced expression of all viral genes. This is accomplished by a combination of alternative splicing (intron retention) and regulated nuclear export of the primary transcript on the RNA processing level and proteolytic cleavage and translational read-through on the post-translational level (reviewed in Refs. 1-4).The genomic organization of all retroviruses is similar (Fig. 1A). The gag-pol open reading frame (ORF) 3 encoding the inner structural proteins (Gag), and the replication enzymes (Pol) is located in the 5Ј half of the transcript and expressed from the unspliced genomic RNA after nuclear export. The gag-pol ORF in all primary retroviral transcripts is defined as an intron through the presence of a preceding 5Ј splice site (ss) in the 5Ј-untranslated region and a functional 3Јss located toward the end of the polymerase ORF (Fig. 1A). To express the glycoproteins (Env), which are encoded in the 3Ј half of the genomic RNA, the gag-pol ORF is removed by a single splice event for subsequent export of the fully spliced RNA (1, 3, 4). This onesplice event strategy cre...

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Mature mRNAs accumulated in the nucleus are neither the molecules in transit to the cytoplasm nor constitute a stockpile for gene expression

Cited by 32 publications

References 42 publications

The translational regulator CPEB1 provides a link between dcp1 bodies and stress granules

The translational regulator CPEB1 provides a link between dcp1 bodies and stress granules

A novel function for Sam68: Enhancement of HIV-1 RNA 3′ end processing

Murine Leukemia Virus Regulates Alternative Splicing through Sequences Upstream of the 5· Splice Site

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